PROJECT SUMMARY/ABSTRACT Vulnerability to relapse despite prolonged abstinence is a principal feature of drug addiction. Decades of research have expanded our understanding of the neural basis of drug addiction, but have yielded few effective treatments that restore top-down control over drug seeking in active and former users. Repeated drug use, but not repeated exposure to natural rewards, results in excess glutamate transmission within corticofugal projections to the striatum in the presence of reward-associated cues and contexts. The dysregulation of glutamate transmission after chronic cocaine use arises in part from enduring changes in fine astrocyte processes that abut active synapses and express glutamate transporters, consistent with a growing literature showing a prominent role for this cell type in learning, memory and synaptic transmission. The goal of this proposal is to explore the mechanism by which the ability of astrocytes to regulate glutamate neurotransmission is altered by addictive drugs. Based upon preliminary data provided in my proposal, I hypothesize that cocaine self-administration leads to a dramatic and enduring shift in the intracellular oxidation state of astrocytes positioned at glutamatergic synapses. This redox shift reduces post-translational glutathionylation of astroglial proteins that are critical for regulating extracellular glutamate concentration, such as GLT-1 and xCT, and proteins regulating astroglial morphology, such as actin and cofilin. The consequent internalization and/or degradation of these proteins results in glial retraction and a reduced capacity to regulate levels of extrasynaptic glutamate, contributing to excessive salience and persistence of drug-related memories. In Aim 1, I describe experiments that will confirm the extent to which these important glial proteins are de- glutathionylated, both after extinction from cocaine seeking and during reinstatement. In Aim 2, I will examine the effects of these redox alterations on glial morphology and surface expression of the principal glutamate uptake transporter GLT-1. Finally, in Aim 3 I will interfere with protein de-glutathionylation specifically in nucleus accumbens astrocytes to determine the extent to which glial protein de-glutathionylation following cocaine exposure affects fine process motility, GLT-1 surface expression, and drug-seeking behavior. As a complementary approach, I will treat cocaine-seeking animals with the antioxidant N-acetylcysteine (NAC) to determine if this therapeutic compound exerts its effects via the same pathway. This work will shed light on the importance of astrocytes in regulating glutamate signaling and contribute to our understanding of the mechanism by which NAC ameliorates reinstated drug seeking.